1. Field of the Invention
The present invention relates generally to the fields of cell culture and virus production. More particularly, it concerns improved methods for the culturing of mammalian cells, infection of those cells with adenovirus and the production of infectious adenovirus particles therefrom.
2. Description of Related Art
Adenoviral vectors, which carry transgenes that can be transcribed and translated to express therapeutic proteins, are currently being evaluated in the clinic for the treatment of a variety of cancer indications, including lung and head and neck cancers. As the clinical trials progress, the demand for clinical grade adenoviral vectors is increasing dramatically. The projected annual demand for a 300 patient clinical trial could reach approximately 1.08×1016 viral particles.
Traditionally, adenoviruses are produced in commercially available tissue culture flasks, “cellfactories,” or RB. Virus infected cells are harvested and subjected to multiple freeze-thaws to release the viruses from the cells in the form of crude cell lysate. The produced crude cell lysate (CCL) is then purified by multiple CsCl gradient ultracentrifugation steps. The typically reported virus yield from 100 single tray cellfactories is about 1×1014 viral particles. Clearly, it becomes unfeasible to produce the required amount of virus using this traditional process. New scaleable and validatable production and purification processes have to be developed to meet the increasing demand.
The purification throughput of CsCl gradient ultracentrifugation is so limited that it cannot meet the demand for adenoviral vectors for gene therapy applications. Therefore, in order to achieve large scale adenoviral vector production, purification methods other than CsCl gradient ultracentrifugation have to be developed. Reports on the chromatographic purification of viruses are very limited, despite the wide application of chromatography for the purification of recombinant proteins. Size exclusion, ion exchange and affinity chromatography have been evaluated for the purification of retroviruses, tick-borne encephalitis virus, and plant viruses with varying degrees of success (Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al., 1978, Smith and Lee, 1978; O'Neil and Balkovic, 1993). Even less research has been done on the chromatographic purification of adenovirus. This lack of research activity may be partially attributable to the existence of the effective, albeit non-scalable, CsCl gradient ultracentrifugation purification method for adenoviruses.
Recently, Huyghe et al. (1996) reported adenoviral vector purification using ion exchange chromatography in conjunction with metal chelate affinity chromatography. Virus purity similar to that from CsCl gradient ultracentrifugation was reported. Unfortunately, only 23% of virus was recovered after the double column purification process. Process factors that contribute to this low virus recovery are the freeze/thaw step utilized by the authors to lyse cells in order to release the virus from the cells and the two column purification procedure.
Clearly, there is a demand for an effective and scaleable method of adenoviral vector production that will result in a high yield of product to meet the ever increasing demand for such products. Recently Blanche et al in WO 98/00524, based on U.S. Ser. No. 60/026,667, describe adenoviral production methods that are useful as descriptive art. PCT publication No. WO 98/00524 and U.S. Ser. No. 60/026,667 are specifically herein incorporated by reference for their description of techniques for production and purification of recombinant adenovirus.